Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

Abstract

The structure, dynamics and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane, and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes, forming nanosize discoidal proteolipid particles, also referred to as native nanodiscs. Here we show, for the first time, that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified with the 2 x 34 kDa-large bacterial cation diffusion facilitator CzcD from Cupriavidus metallidurans CH34. Keywords membrane protein; nanodiscs; styrene maleic acid; NMR spectroscopy; solid-state NMR Integral membrane proteins play essential roles in numerous cellular processes. The properties of the surrounding lipid environment are crucial to their structure, dynamics and function. However, structural and biophysical characterizations by most biophysical techniques generally require extraction and purification from the native membrane. Major concerns when working with detergent-solubilized membrane proteins are related to the fact that functionally and structurally important lipids are often stripped away, and that the properties of the detergent micelle differ significantly from the planar lipid bilayer environment. An increasing number of cases of distortions of membrane protein structures in detergents have been reported.[1,2] These known problems with detergents have triggered the development of alternative non-micellar systems, such as amphiphilic polymers (amphipols), bicelles, or nanolipoprotein particles, i.e. a lipid patch surrounded by a membrane-scaffold protein. [3][4][5] A particularly interesting recent approach is the use of the beate.bersch@ibs.fr, paul.schanda@ibs.fr. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts styrene/maleic acid co-polymer (SMA), which is able to self-insert into the lipid bilayer, and to extract membrane proteins directly from the membrane, forming nanosize disc-shaped particles. [5][6][7] SMA has been successfully used to solubilize membrane proteins from reconstituted proteoliposomes [8,9], and also from native cellular membranes, forming socalled native nanodiscs. [7,[10][11][12][13][14] The stability and activity of proteins in SMA-bounded nanodiscs have been shown to be significantly higher than in detergent micelles in several instances, and some cases were reported where the stability was even higher than in the native membrane [15]. These nanodiscs have been used in functional tests, ligand binding studies, electron-microscopy, EPR and other spectroscopic characterizations (reviewed in...

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…The potential of NMR spectroscopy to resolve integral membrane proteins in SMALPs is demonstrated by a recent study of the bacterial zinc diffusion facilitator CzcD [32]. This 34 kDa protein could be similarly solubilized with either XIRAN 25010 and 30010, retaining a single layer of bound lipid, and purifies well with Strep tag.…”

Retracted Article: Polymer nanodiscs and macro-nanodiscs of a varying lipid composition

“…4–7 Recent studies reported various physicochemical properties of lipid bilayers encapuslated by a polymer in naodiscs. 8–15 Because of the inexpensive production and high stability, polymer based nanodiscs are increasingly utilized in the extraction and structural studies of membrane proteins 16–18 , and also in nanobiotechnological applications including drug delivery 19, 20 . In particular, a recently introduced small molecular weight SMA-EA polymer dramatically enhanced the previously reported maximum nanodisc size limit of ~20 nm to ~60 nm diameter and also exhibited better stability to low pH and high concentrations of divalent metal ions.…”

Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

“…[6] Some of the important discoveries of the last decade in P450 related research have foundations on those pioneering observations. [108][109][110][111][112][113][114][115][116][117][118][119][120][121] We also expect the macro-nanodiscs to be highly valuable for studies using cryoEM and diffraction experiments. [107] Forty-years later, those cautious speculations were ultimately and quantitatively demonstrated by our group using sophisticated tools.…”